1. Field of the Invention
The present invention relates generally to measurement of fluid samples and, more particularly, to optical measurement of such samples in a photometric flow cell. The invention is particularly suited for use in a liquid chromatographic system where a stream of successive fluid sample fractions eluted from a chromatrographic column is flowed through a photometric flow cell for analysis of individual sample fractions.
2. Description of the Prior Art
Double beam photometers have long been used in fluid flow systems to optically measure fluid samples. In one common form, such photometers comprise a flow cell body having a pair of light transmissive passageways--a sample passageway configured to receive a fluid sample and a reference passageway configured to receive a reference fluid or no fluid at all. Light from a source is split into a sample beam and a reference beam and passed through the corresponding sample and reference passageways and hence through any sample or reference fluid therein. A light detector intercepts light emerging from the respective passageways to derive a ratio measurement of sample and reference beam energy and hence a measure of a characteristic (usually absorbance) of the sample. A principal advantage of a double beam system is its inherent ability to reject common mode noise effects in the two paths, and hence to generate an output signal exhibiting a high common mode rejection ratio.
In this pressure liquid chromatography (HPLC), double beam flow cell photometers have been adapted to receive and optically measure a stream of successive fluid sample fractions eluted from a chromatographic column. The sample fractions (carried in a solvent matrix) are typically minute in volume (e.g. 100 .mu.l-1.0 ml), closely spaced, low in concentration, and are flowed at high pressure up to as high as about 40 atmospheres in the flow cell.
In designing double beam flow cells for HPLC applications, configurations are desired which measure sample fractions with maximum resolution and sensitivity. Resolution is an indication of the system's ability to distinguish measurements of successive, closely spaced flowing samples, i.e. to distinguish the "peaks" generated in the photometer output optical signal representative of such samples. Sensitivity is an indication of the minimum concentration or amount of sample which must be present to be distinguished as sample from the baseline noise of the photometer output signal. In these respects a common prior flow cell configuration comprises sample and reference passageways identical in size and shape. Apparently, identical sample and reference passageways were felt to be desirable to provide a symmetry or balance in the optics geometry of the flow cell to establish adequate common mode rejection and resolution for the system
As prior flow cell designs have been perfected, so-called "shot"noise in the photometer output signal has become a more significant factor. Shot noise is a high and low frequency noise signal superimposed on the output optical baseline signal of the photometer which is believed to represent statistical variations in light beam intensity. The effect of the shot noise signal is to decrease the sensitivity of the photometer. By decreasing system sensitivity, shot noise impairs the system's ability to measure samples of low concentrations. In effect, then, the performance of the prior flow cells, though improved, has become limited by shot noise. One possible approach for contending with shot noise is to increase the light energy level in the sample passageway by increasing the passageway diameter. While such effects a shot noise reduction, it is at the expense of a substantial decrease in system resolution.
Accordingly, a need exists for a double beam photometer for receiving and measuring fluid samples without the drawbacks of the prior art. The present invention meets these needs.